Project Summary/Abstract The inner ear is the primary sensory organ mediating hearing (auditory) and balance (vestibular) function in all vertebrates. Common to both the auditory and vestibular sensory epithelia are sensory hair cells, which are acutely sensitive to acoustic trauma. Loss of hair cells in the mammalian inner ear is irreversible and causes hearing impairment, deafness and balance dysfunction. In contrast to mammals, fish not only produce new hair cells throughout life but also have the innate capacity to regenerate functional hair cells. Using zebrafish as a regenerative animal model, this proposal is focused on the formation and regulation of long-lived hair cell precursors and the genetics of nascent hair cell reinnervation. The long-term goal of this proposal is to provide a molecular and genetic understanding of hair cell renewal in zebrafish and use this information to develop novel strategies that force hair cell regeneration and reinnervation in mammals. Specific Aim 1 is focused on the formation and regulation of postmitotic hair cell precursors that mediate non-mitotic hair cell regeneration in zebrafish. Using a newly identified strain of zebrafish, we show that Notch mRNA is expressed in the postembryonic sensory epithelium and that inhibitors of 3-secretase activity induce precocious hair cell formation in vivo. The goals of this Aim are to (a) determine the role of Notch signaling in regulating precursor differentiation, (b) determine the function of postmitotic precursors during growth and regeneration in the sensory epithelium, (c) identify the mitotic source of long-lived hair cell precursors, and (d) determine the genetic basis of non-mitotic hair cell regeneration in zebrafish. Specific Aim 2 will identify genes required for neuronal reinnervation of nascent sensory hair cells. Restoration of auditory function requires the inextricably linked processes of cell renewal and reconnection to the central nervous system. However, hair cell regeneration and reinnervation can be dissociated in vivo using molecular genetics and mutant identification in zebrafish. By exploiting simple visual and behavioral assays we will focus on (i) zebrafish mutants that do not regenerate hair cells and (ii) mutants that regenerate, but do not reinnervate nascent hair cells. Cloning of the affected loci in these mutants will identify novel genes, or known genes with novel functions, required for new hair cell formation and reestablishment of synaptic connectivity. PUBLIC HEALTH RELEVANCE: 7. Project Narrative Hearing loss is the single most common individual disability in the veteran population. Nearly 60% of soldiers exposed to explosive blasts suffer permanent hearing loss and 28% of troops returning from a war zone have diminished hearing. Currently, more than 800,000 veterans are receiving disability compensation for service-connected hearing disorders, which is expected to grow by 18% a year with payments totaling $1.1 billion annually by 2011. Results of these proposed studies will provide new insight to the innate mechanisms of sensory hair cell regeneration and reinnervation. Since regeneration does not occur in mammals, this information is necessary to direct current research efforts and design novel strategies aimed toward ameliorating or reversing the effects of hearing loss in humans.